1. Field of the Invention
This invention relates to a composition and method for preparing the composition which includes the removal mycotoxins and enzymatic hydrolysis to form bioactive peptides from agricultural products that are contaminated with mycotoxins.
2. Description of the Related Art
Mycotoxins are naturally occurring substances produced by certain species of fungi including, for example, Aspergillus sp., Fusarium sp., Penicillium sp. These fungi commonly grow on and infest plant materials such as grains, oilseeds, and grasses. They are most often produced in the field under conditions of environmental stress on the plant (e.g. heat, insects, and drought). Mycotoxins include aflatoxins, ochartoxins, zearalenones, T-2 toxin, HT-2 toxin, diacetoxyscipenol, monoacetoxyscripenol, neosolaniol, nicalenol, deoxynivalenol, 3-acetaldeoxynivalenol, T-2 tetraol, scripentriol, fusarenon, crotoxin, stratoxin H, etc. Aflatoxins are mycotoxins that present remarkable toxicity and hepatocarcinogenicity. Aflatoxins can cause diverse toxic effects on virtually all organs, eventually leading to the development of cancerous tumors capable of spreading throughout the entire body. There are four major aflatoxins: AfB1, AfB2, AfG1 and AfG2, that contaminate crops, with AfB1 and AfG1 having greater toxic potential than aflatoxins AfB2 and AfG2. The International Agency for Research on Cancer has particularly noted that the major forms AfB1 and AfG1, as potent carcinogens, linked primarily to cancer of the liver. Thus, the amount of aflatoxin allowed in human and animal food is regulated by State and Federal agencies. Fumonisin B1 is a mycotoxin that occurs almost exclusively on corn and can cause toxic effects in horses and swine. Fumonisin B1 has been linked to esophageal cancer in humans and has been shown to be a caner initiator and promoter in rodents. Tricothecenes such as for example T-2 toxin, deoxynibvalenol or vomitoxin; ergot, zearolenone, cyclopiazonic acid, patulin, ochartocin A, and secalonic acid D are mycotoxins that can negatively affect impact human and animal health due to their diverse toxic effects. The toxic effects caused by these mycotoxins may be classified as acute or chronic, depending on the level and duration of mycotoxin exposure and species sensitivity.
Virtually all animals in the food chain can be affected by exposure to contaminated food and feed, including humans, who can be exposed directly to toxins through grain handling and consumption or directly through consumption of an unmetabolized parent compound or toxic metabolite products in contaminated meat or livestock products such as milk and cheese. As a result, mycotoxin contamination of agricultural commodities such as corn, wheat, rye, rice, barley, oats, peanuts, pecans, soybeans, cottonseed, apples, grapes, alfalfa, clover, sorghum and fescue grass forages, can result in severe economic loss at all levels of food production such as cost of preharvest prevention, post-harvest treatment, productivity and increased loss of livestock, health care costs, etc.
Oil processing conditions are chosen to optimize the maximum amount of oil extraction with little regard for protein. Using peanut meal, as an example, approximately 97% of the total protein is contained in the two globulins, arachin and conarachin (Basha, S. M. M. Identification of cultivar differences in seed polypeptide composition of peanuts by two-dimensional polyacrylamide gel electrophoresis Plant Physiol. 1979, 63, 301-306). Defatted peanut meal protein content is highly dependent on the type of oil extraction technique used (Basha, S. M. M.; Cherry, J. P. Composition, solubility, and gel electrophoretic properties of proteins isolated from Florunner peanut seeds J. Agric. Food Chem. 1976, 24, 359-365.). Defatted peanut meal can be prepared by hydraulic pressing, screw pressing, solvent (hexane) extraction or pre-pressing followed by solvent extraction (McWatters, K. H.; Cherry, J. P. Potential food uses of peanut seed proteins In Peanut science and technology; Pattee, H. E.; Young, C. T., Eds.; American Peanut Research and Education Society: Texas, 1982; pp 689-736; Cherry, J. P. Peanut protein and product functionality, J. Am. Oil Chem. Soc. 1990, 67, (5), 293-301).
In the early 1900's, the non-food grade peanut meal by-product of oil pressing was sold as cattle feed at about thirty-five dollars per ton (Johns, C. 0.; Jones, D. B. The proteins of the peanut, Arachis hypogaea. I. The globulins arachin and conarachin. J. Biol. Chem. 1916, 28, (1), 77-87.). Aflatoxin contaminated peanut meal is sold as animal feed at approximately one hundred seventy-five dollars per ton if the aflatoxin contamination is between 20 to 300 parts per billion (ppb). If the peanut meal has less than 20 ppb, it can be sold as dairy cattle feed at a premium price of approximately two-hundred ten dollars per ton. Highly contaminated peanut meal, greater than 300 ppb, can be sold as fertilizer or mushroom compost at approximately ninety-five dollars per ton (prices are approximate and fluctuate).
Aflatoxins are toxic, carcinogenic compounds which are produced by the fungi Aspergillus flavus Link and Aspergillus parasiticus Speare (Monteiro, P. V.; Prakash, V. Effect of proteases on arachin, conarachin-I, and conarachin-II from peanut (Arachis-hypogaea L). J. Agric. Food Chem. 1994, 42, (2), 268-273.). There are four major naturally occurring aflatoxins, aflatoxin B1, B2, G1, and G2 (Ramos, A. J.; FinkGremmels, J.; Hernandez, E. Prevention of toxic effects of mycotoxins by means of nonnutritive adsorbent compounds. J. Food Prot. 1996, 59, (6), 631-641.). These four compounds are distinguished by their fluorescence color (B=blue; G=green) and their relative chromatographic mobility (McLean, M.; Dutton, M. F. Cellular interactions and metabolism of aflatoxin—an update. Pharmacology & Therapeutics 1995, 65, (2), 163-192.). Aspergillus flavus only produces aflatoxin B1 and B2). Aflatoxin M1, found in milk as a metabolite of aflatoxin in cattle feed, is a hydroxylated form of aflatoxin B1.
Most peanut oil is a product of aflatoxin-contaminated peanuts. After the oil is extracted, the aflatoxin remains in the by-product, peanut meal (note: aflatoxin, like all solids in peanuts, are concentrated in the peanut meal after the removal of the oil). The aflatoxin level in the peanut meal must be quantified before it can be sold as animal feed, see Table 1 below. The susceptibility of animals to aflatoxicosis depends upon 1) their ability to activate aflatoxin B1 to aflatoxin Bi-8,9-epoxide and 2) their ability to convert aflatoxins to form glucuronide or sulphate conjugation products to be excreted (Roebuck, B. D.; Wogan, G. N. Species comparison of in-vitro metabolism of aflatoxin-B1. Proc. Am. Assoc. Cancer Research 1974, 15, (MAR), 68-68).
TABLE 1Action levels for aflatoxin to control contamination inhuman food and animal feed, as determined by the FDA (61).Action Level(ppb)CommodityPeanuts and peanut products20Pistachio Nuts20Brazil Nuts20Human Foods20Milk0.5(aflatoxinM1)Animal FeedPeanut products intended for finishing beef cattle300Peanut products intended for finishing swine of 100 pounds200or greaterPeanut products intended for breeding beef cattle, breeding100swine, or Mature poultryPeanut products intended for immature animals20Peanut products intended for dairy animals, for animal20species or uses Not specified above, or when theintended use is not knownCurrent research for detoxifying or inactivating aflatoxins to protect food and animal feed from the toxic effects include irradiation, solvent extraction, density segregation, microbial inactivation, ammoniation, adsorptive materials, and thermal inactivation(Phillips, T. D.; Clement, B. A.; Park, D. L. Approaches to reduction of aflatoxins in foods and feeds. In The toxicology of aflatoxins; Eaton, D. L., Groopman, J. D., Eds. Academic Press: New York, 1994; pp 383-406). Adsorptive materials, or sequestering agents, such as activated charcoal, bentonite and aluminosilicates can be mixed into contaminated animal feed to bind aflatoxins (note that binding occurs upon consumption, i.e. in the GI tracts of livestock), enabling them to pass through the animal gastrointestinal tract, guarding against aflatoxicosis (Ramos, A. J.; FinkGremmels, J.; Hernandez, E. Prevention of toxic effects of mycotoxins by means of nonnutritive adsorbent compounds. J. Food Prot. 1996, 59, (6), 631-641; Huwig, A.; Freimund, S.; Kappeli, O.; Dutler, H. Mycotoxin detoxication of animal feed by different adsorbents. Toxicol. Lett. 2001, 122, (2), 179-188). The ideal toxin-binder should not dissociate internally and should be expelled in the animal feces (Diaz et al., Mycopathologia, Volume 156, 223-226, 2002)). Zeolites, hydrated sodium calcium aluminosilicates (HSCAS) and aluminosilicate-containing clays are the most commonly studied mycotoxin adsorbents. Aluminosilicate clays are generally recognized as safe (GRAS) and the U.S. FDA approved their use as anticaking agents in animal feed up to approximately 2% dry weight basis under title 21, sections 582.2727 and 582.2729 in the Code of Federal Regulations (United States Food and Drug Administration. Code of Federal Regulations—Part 582 Substances Generally Recognized as Safe. http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm (accessed Aug. 29, 2008)). In vitro aflatoxin binding experiments may not give an accurate prediction of in vivo animal protection.
Activated charcoal is a non-soluble powder formed by pyrolysis of organic materials (Huwig, A.; Freimund, S.; Kappeli, O.; Dutler, H. Mycotoxin detoxication of animal feed by different adsorbents. Toxicol. Lett. 2001, 122, (2), 179-188). This substance is very porous with a high surface area which provides for adsorption of numerous toxic materials, including aflatoxins, making them unavailable for gastrointestinal absorption (Ramos, A. J.; FinkGremmels, J.; Hernandez, E. Prevention of toxic effects of mycotoxins by means of nonnutritive adsorbent compounds. J. Food Prot. 1996, 59, (6), 631-641). Historically, activated charcoal has been used in the medical field for treating poisoning and drug overdoses. Although activated charcoal is odorless, tasteless and non-toxic, it will absorb nutrients, vitamins and minerals, making it unsuitable for use in animal feed.
Yano et al. (U.S. Pat. No. 4,055,674) disclose a method for removal of aflatoxin from materials using a mixed solvent system of liquid dimethyl ether and water. The method reduces the aflatoxin content to 15 ppb or less.
Bentonite, a layered crystalline microstructure comprised primarily of montmorillonite, can also be used to adsorb molecules such as aflatoxins (Ramos, A. J.; FinkGremmels, J.; Hernandez, E. Prevention of toxic effects of mycotoxins by means of nonnutritive adsorbent compounds. J. Food Prot. 1996, 59, (6), 631-641). This clay substance is GRAS approved as a direct food additive and is currently used to remove the protein in white wine processing and to sequester aflatoxins in animal feed.
HSCAS has positive charge deficiencies which create the potential for adsorbing cationic compounds and positively charged molecules, such as aflatoxins (Ramos, A. J.; FinkGremmels, J.; Hernandez, E. Prevention of toxic effects of mycotoxins by means of nonnutritive adsorbent compounds. J. Food Prot. 1996, 59, (6), 631-641). Similarly, zeolites are very porous with a high surface area and a high cation exchange capacity (Huwig, A.; Freimund, S.; Kappeli, O.; Dutler, H. Mycotoxin detoxification of animal feed by different adsorbents. Toxicol. Lett. 2001, 122, (2), 179-188). The surface is polar and binds polar mycotoxins. Zeolite is GRAS and the FDA approves its use as a feed additive and an anti-caking agent (United States Food and Drug Administration. Code of Federal Regulations—Part 582 Substances Generally Recognized as Safe. http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm (accessed Aug. 29, 2008)). This substance is currently used by beef and dairy cattle, broiler, commercial egg, swine, sheep and turkey producers (ZEO, Inc. Zar-Min Benefits Proven in Research. http://www.zeoinc.com/zar-min.html (accessed Sep. 24, 2008)).
Proteins play a vital role in food functionality and quality, in addition to fulfilling basic nutritional needs. Protein functionality within a food system is highly dependent on solubility and degree of denaturation. Enzymatic hydrolysis of proteins is an established method of generating peptides that have been shown to enhance functional properties such as foaming, emulsification, and solubility, as well as improving nutritional quality (Adler-Nissen, J. Determination of the degree of hydrolysis of food proteinhydrolysates by trinitrobenzenesulfonic acid. J. Agric. Food Chem. 1979, 27, (6), 1256-1262).
Alcalase, pepsin and Flavourzyme are all water soluble, food-grade, commercially available enzymes. These proteases have been well studied and are used to enhance protein functionality in both commercial food and research applications. Bioactive peptides are short-chain amino acids which exhibit specific biological effects, such as antioxidant capacity, upon consumption (Korhonen and Pihlanto, Current Pharmaceutical Design, Volume 9 (16), 1297-1308, 2003). Bioactive peptides can be generated outside the body through hydrolysis, and then consumed, or digested and released naturally inside the body. Currently established sources of bioactive peptides include: chickpea (Clemente et al., J. Agric. Food Chem., Volume 47 (9), 3776-3781, 2007), sunflower (Megias et al., J. Agric. Food Chem., Volume 55 (16), 6509-6514, 2007), corn (Li et al., J. Sci. Food Agric., Volume 88 (9), 1660-1666, 2008), canola (Cumby et al., Food Chem., Volume 102 (1), 144-148,2008), soybean, wheat, rice, barley, and buckwheat (Wang and Mejia, Comprehensive Reviews in Food Science and Food Safety, Volume 4, 63-78, 2005). Recent studies have suggested that peanut protein hydrolysates could be used as a natural antioxidant. The effect of roasting time coupled with enzymatic hydrolysis of roasted defatted peanut seeds on antioxidant capacity was studied (Hwang et al., Comprehensive Reviews in Food Science and Food Safety, Volume 34, 639-647, 2001). It was concluded that antioxidant capacity increased with roasting time from 0 to 60 min at 180° C. and increased further when hydrolyzed with either Esperase or Neutrase. More recently, Chen et al. (J. Sci. Food Agric., Volume 87 (2), 357-362, 2007) reported the antioxidant capacities of peanut protein hydrolysates by measuring the inhibition of linoleic acid autoxidation, scavenging effect on free radicals, reducing power and inhibition of liver lipid autoxidation. Peanut protein hydrolyzed with Alcalase had increased antioxidant capacity over unhydrolyzed peanut protein, but slightly less antioxidant capacity than butylated hydroxytoluene, a synthetic antioxidant (w/v basis) (Chen et al, 2007,supra).
While various systems have been developed for preparing bioactive peptides from other plant materials, there still remains a need in the art for a method for producing a high protein peanut oil by-product that has at least FDA approved levels of aflatoxin in a human food product and contains bioactive peptides. The present invention, different from prior art systems, provides such a method and a nutritional peanut meal human food product made by the novel method.